Adenovirus (Ad) represent a significant causes of human respiratory, gastrointestinal and ocular infections; however, replication-defective forms of Ad are currently in use in clinical trials for gene therapy. Despite some success with this approach, a lack of knowledge of how Ad recognizes host cell co-receptors (alphav integrins) and how alphav integrins promote virus uptake has prevented optimal use of Ad vectors in the clinic. This proposal seeks to define the precise integrin binding events and intracellular signaling pathways involved in adenovirus entry into host cells. The goals of this proposal are to 1) determine the three-dimensional architecture of Ad and the kinetics of alphav integrin binding, 2) to characterize the mechanism of Ad-mediated endosome disruption and the role of alphav integrins in this process, 3) to define the signaling molecules that regulate Ad internalization and 4) to exploit cell signaling pathways to enhance Ad-mediated gene delivery in vitro and in vivo. Cryoelectron microscopy and kinetic analyses will be used to define the structural elements responsible for alphav integrin association with different Ad serotypes and extracellular matrix proteins. A panel of epithelial cells lines expressing modified forms of alphav integrins will be used to identify the precise sequences and mechanisms involved in Ad-mediated endosome disruption, a key step required for virus penetration into the cell. Biochemical and molecular genetic approaches will be used to investigate the role of signaling molecules in virus internalization (endocytosis). Finally, a modified adenovirus vector with the capacity to trigger multiple cell signaling pathways will be used to enhance gene delivery to neovascular tissue and solid tumors in vivo. If successful, these studies have the potential to increase our understanding of several fundamental cell and molecular biological processes as well as optimize the use of Ad vectors.